Serotonergic neurotransmission plays a critical role in many physiological functions. Nevertheless, little is known about the properties of serotonergic synaptic vesicles. Since it is difficult to purify either serotonergic neurons or their synaptic vesicles from the brain, we have studied the 5-HT-containing secretory vesicles of parafollicular (PF) cells of the thyroid as a model. PF vesicles are unique in that they resemble both the trans-Golgi network-derived vesicles of regulated secretion (in containing peptides) and the endosome-derived synaptic vesicles (in containing a recycling membrane and a small molecular transmitter). Unlike other secretory vesicles, PF vesicles are not constitutively acidic, but acidify when PF cells are exposed to a secretagogue, i.e., increased (Ca2+)e. Acidification results from the stimulus-coupled gating of a vesicular Cl- channel that permits influx or H+. Gating of the Cl- channel is induced by its phosphorylation. The resulting delta pH maximizes the vesicular uptake of 5-HT and thus contributes to the level of secretion. Activation of a G protein-coupled Ca2+ receptor (CaR) on the plasmalemma of PF cells regulates downstream effectors (including: phospholipase C, NO synthase and guanylyl cyclase) that lead to vesicle acidification. We now propose to determine the extent to which this transduction pathway is shared by the secretion process and the degree to which the secretory vesicles of PF cells are similar to the synaptic vesicles of serotonergic neurons. Aim 1 is to determine the mechanism underlying CaR-induced gating of the vesicles of PF cells, by focusing on the relationship between vesicle acidification and secretion as measured by quantal release of 5-HT from PF cells. The interaction of CaR with the voltage-gated Ca2+ channels present in PF cells will be studied using patch-clamp recordings, accompanied by Ca2+ imaging. Aim 2 will be to compare properties of PF vesicles with those of synaptic vesicles of serotonergic neurons in situ and immortalized neuronal serotonergic cell line RN46A. We will examine whether these synaptic vesicles exhibit stimulus-induced acidification, and whether CaR participates in regulating the acidity of serotonergic synaptic vesicles. Secretory and synaptic vesicles that regulate internal pH in response to plasmalemmal stimulation are novel and, when understood, are likely to provide insight into serotonergic neurotransmission, as well as the drugs that affect it.